SAKMANN AND NEHER INVENT PATCH-CLAMPING Flashcards
A method to measure the behavior of a single ion channel
SAKMANN AND NEHER
PATCH-CLAMPING
Erwin Neher and Bert Sakmann invented a new technique called patch
clamping, which allowed them to study the
behavior of a single ion channel
1991 Nobel Prize in Medicine or Physiology
Neher and
Sakmann
The ion channel is
inserted in the
membrane that is
in the lumen of the electrode,
However, whether
open for a longer or shorter period, the conductance of the Na channel (the amount of current passed
during any instant in time) is always the same. T/F
True
individual channel opens and closes
very rapidly
channels have ___ conductances
unitary
If the same depolarizing pulse was presented a
second time, or a third or fourth time, the same inward current pattern was evoked. This pattern
of current flowing through the entire axon is called the
macroscopic current
patterns of inward current evoked in a single Na channel, called the
microscopic current,
The opening and closing of an individual channel was variable because the behavior of any
channel is
probabilistic.
What the membrane potential does is to change the ___that a
channel will open.
probability
In the case of the Na channel, depolarization increases the probability that the
activation gate will open.
hyperpolarization
increases the probability of
closing the Na activation gate
depolarization increases the probability that the inactivation gate will
close
hyperpolarization
increases the probability that the inactivation gate will
open
How then do we reconcile the stereotyped current response of the squid giant axon to a
depolarization of -10 mV, the shape of the macroscopic current, with the highly variable
response of an individual Na channel, the microscopic current, to exactly the same
depolarization?
squid giant
axon during voltage clamp shows the summated (or averaged) currents through many ion
channels in the entire membrane of the axon rather than the current through a single channel.
was simple to calculate
the probability of a Na channel opening as a function of
membrane potential
function of membrane potential. That function is shown in Fig. 4E, and has a \_\_\_shape
sigmoidal
inward currents are
shown as
downward deflections
Current through a single
channel is called the
microscopic
current
The sum of many such microscopic currents shows that most channels open in the initial\_\_\_after which the probability of channel openings diminishes because of channel inactivation.
1-2 ms,
The macroscopic
current resulting from the current
flowing through all of the Na channels
while
voltage clamping the entire axon
is very similar
to the summed microscopic current in C.
macroscopic current
The probability of Na channel
opening depends on the
membrane
potential,
Notice that the
probability is virtually zero (few or no
channels open) at
-60 to -80 mV
probability of channel opening is about
80% even when the membrane is
depolarized to
+40 mV
Depolarizing steps cause outward currents which are seen as
(upward
deflections
Notice that the K channel opens with different
latencies to the depolarization and that the channels
stay open for the length of the
depolarization,
the probability-membrane
potential function is similar to both the K
conductance-membrane potential function and to the
Na conductance-membrane potential function
T/F
T
Strong hyperpolarization virtually ensures that
52
activation gates are closed, since the probability of a Na or K channel being open at ____
is just about zero (
-70-80 mV
the open probability increases
sharply with depolarization at these values
-40 to 0 mV
max probability at
+30 mV.
What is somewhat surprising however is that the maximum open probability is not 100%
but rather only
70-80%
hyperpolarization
guarantees gate closure whereas depolarization only makes gate opening more likely. T/F
T
he probability that the channel can pass Na+ ions decreases after the
depolarization
been present for a period of time due to the closure of the inactivation gate
The delayed closure can be appreciated from the records in Fig. 2 and Fig. 4B, since there were
no channel openings after the first
first 8.0 msec or so of the depolarizing pulse.
It has been known that if a nerve axon is hyperpolarized for a period of time, an
action potential is often set off when the hyperpolarization is released and the cell quickly
brought back to rest; this is called
anode break excitation or rebound excitation.
explanation for rebound excitation is that
hyperpolarization of the membrane increases
the number of Na channels available for opening in response to depolarization
why the states of the activation and
inactivation gates are “probabilistic”.
This “flickering” between states
even when the membrane potential is not changing,
The larger number of Na channels open when the membrane potential was brought back to
rest allowed a correspondingly
larger influx of Na+ ions
two features of axons that determine velocity
axon diameter; and 2) myelination
larger the axon diameter, the ___ the conduction velocity
faster
The longer the advance of the current, the ____ the action potential propagates
faster
How far the local
current spreads down the axon depends upon two features that act as resistances to current flow. They
are;
membrane resistance, and 2) the internal resistance of the axon
The membrane resistance is determined simply by the
number of open ion channels in the
membrane.
(K+ channels that set the resting potential, that
is, the K+ channels that are NOT gated by voltage but are always open.)
refers to how difficult it is for current to flow down the axon.
internal resistance
The internal resistance refers to how difficult it is for current to flow down the axon. Obviously,
the larger the diameter, the ___resistance there is to flow.
less
larger axons have a ____ internal resistance, and hence it is easier for current to flow down the axon and depolarize long segments
of the axon
lower
the larger the axonal diameter, the ___the membrane resistance and the
more current that leaks out along the axon
smaller
membrane resistance acts to oppose the
effects of
internal resistance
Thus decreasing membrane resistance ___the time it takes for the axon to reach threshold
lengthens
slow conduction velocity
The question is, which of the two features dominates, internal resistance or
membrane resistance?
internal resistance
Membrane resistance is
determined by the
axonal
circumference
internal
resistance is determined by
area of
the axon.
changes as the square of the radius
and thus changes faster with
diameter
Internal resistance
Circumference of circle
2 x pi x r
area of a circle
pi x r^2
are those that are voltage insensitive, i.e., are not influenced by voltage
Passive
properties
Passive
properties are
conduction velocity, membrane resistance, internal
resistance and capacitance
active properties that are voltage sensitive,
such as
voltage gated Na+ and K+ channels
electrical component that simply is an insulator that separates and thereby stores charges
capacitor
The quantitative feature of a capacitor is called its
capacity.
simply the amount of charge
required to change the potential across the capacitor by 1 millivolt.
capacity.
Capacity =
Capacity =
Q/ V, where Q is charge (coulombs) and V is voltage.
So the passive properties are determined by three principal features:
1) membrane resistance; 2)
membrane capacitance and 3) internal resistance.
each
patch is connected to the next patch of membrane through the
internal resistance (ri).
other words, the charge across capacitor in patch 1 experiences the
greatest change (the largest depolarization).
Each successive patch of membrane
receives less current than the previous patch because; 1
1) much of the current has already been used to
depolarize the set of capacitors closest to the current source; and 2) some of the current has leaked out in
the initial patches through open channels that form the membrane resistance.
If the depolarization of the membrane potential at patch 1 is sufficiently large, the signal is
prevented from dying out by
active processes that are voltage dependent
nature has done in vertebrates is to add
myelin sheaths to smaller axons
Since all membranes
are made of lipids, the fatty wrapping, called myelin, forms an
insulation, just
The insulation prevents current from leaking out along the length of the insulated portion, i.e., it
massively increases
membrane resistance
The entire length of the axon is not insulated without
interruption. Rather, there are bare spots located periodically along the axon called
nodes of Ranvier.
All voltage gated Na+ and K+ channels are densely packed at the
nodes of Ranvier.
What
happens, in essence, is that the action potential in a myelinated axon
skips from node to node
This type of conduction used by myelinated axons is called
saltatory conduction
saltatory conduction
Latin saltare,
hop or leap
The largest myelinated axons in our nervous system have diameters of only
20 micrometers (recall that the diameter of the squid giant axon is 800-1000 micrometers
an
unmyelinated fiber would have to be___ in diameter to conduct as fast as the most rapid myelinated
fibers.
4 mm
Multiple sclerosis (abbreviated MS) is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are
damaged or degenerate
The name multiple
sclerosis refers to scars (scleroses—better known as plaques or lesions) particularly in the
white matter
of the brain and spinal cord, which is mainly composed of myelin
In MS, the body’s own immune
system attacks
oligodendrocytes,
there are non-voltage gated K+ channels but no voltage-gated
Na+ or K+ channels under the
myelin sheaths (
conduction
velocity in vertebrates is determined by both
myelination and axon diameter.
Our unmyelinated axons have conduction velocities of around
1 meter/sec,
myelinated nerve fibers conduct from
10-120 meters/sec
The recording electrodes are on the surface of the nerve and record the
summated action potentials of all the fibers as they travel along the nerve. This recording is
compound action potential
compound action potential has three major components
first
component evoked reflects the arrival of the fastest (largest) myelinated axons and is called the A
component.
smaller myelinated axons and is called the B
component.
slowest axons are unmyelinated
touch, joint position,
muscle stretch and sharp pain
A component
slow pain and temperature
receptors,
C component.
slowest axons are unmyelinated
The fastest fibers are called
Aa fibers
next fastest are
Ab fibers
slowest A fibers are
the
Ag fibers
The nerves of the
peripheral nervous system are
mixed nerves;
action potentials in the
axons of motor neurons move
orthodromically) toward their muscles in the thumb
action potentials in the axons of the sensory fibers move
backward
antidromically
the conduction velocity of both sensory and motor myelinated fibers is
slowed down, or even abolished, due to demyelination, whereas the conduction velocity of
unmyelinated fibers is normal
MS
selective degeneration of motor neurons; sensory neurons
are not affected. The selective pathology and death of motorneurons is detectable by observing
decreased, slowed, or jittery motor components (at the base of the thumb) with normal sensory
components (at the fingertips since no motoneurons are present in the fingertips).
ALS also called Lou Gehrig’s
disease)
